Project Summary/Abstract Heterotrimeric G proteins are important molecular switches that play critical roles in mediating cellular responses to a variety of signals. Mutations in G proteins have been implicated in many human diseases that include heart diseases, endocrine diseases, and cancer. Thus, a thorough understanding of the functions of G proteins and their mechanisms of regulation is very important. The goal of this project is to study novel functions of G alpha protein and its mechanisms of regulation, with a particular focus on its function in intracellular organelles. Most studies on G proteins have focused on their canonical functions on plasma membrane. However, G proteins are also found in intra-cellular organelles, and accumulating evidence indicates that intracellular G proteins can exert important signaling functions. In our preliminary studies, we find in yeast that a pool of Gpa2, a G alpha protein, is present in mitochondria and localization of Gpa2 to mitochondria is regulated by nutrient status. Interestingly, Gpa2 also undergoes nutrient status-dependent phosphorylation and blocking this phosphorylation event impairs how cells respond to nutrient deprivation. Our objectives are to study the role of phosphorylation in regulating Gpa2 and to elucidate the novel mitochondrial functions of Gpa2. To achieve our objectives, two specific aims are proposed. Aim 1: What are the functional consequences of Gpa2 phosphorylation? To elucidate the role of Gpa2 phosphorylation, we will identify its precise phosphorylation sites, generate mutants that either block or mimic phosphorylation, and examine their signaling behaviors. We will also identify enzymes (kinases and phosphatases) that control Gpa2 phosphorylation and examine the effects of their disruption on Gpa2 signaling. To establish the mechanistic basis for any observed effects of Gpa2 phosphorylation on signaling, we will determine how altering Gpa2 phosphorylation impacts Gpa2 properties that include its subcellular localization, its interaction with the activator (Gpr1) and the effector (adenylyl cyclase), as well as its GTPase activity. Aim 2: What is the role of Gpa2 in mitochondria? Adenylyl cyclase Cyr1, an important effector of Gpa2, is present in mitochondria and its activation in mitochondria is important for cells to adapt to changes in the nutrient status. We hypothesize that a function of Gpa2 in mitochondria is to regulate the activity of Cyr1. To test this, we will purify mitochondria from cells with or without Gpa2 and examine the activity of mitochondrial Cyr1. To identify novel Gpa2 effectors in mitochondria, we will purify mitochondrial Gpa2 complex and sequence proteins that specifically interact with Gpa2 in mitochondria. The novel Gpa2-interacting proteins will be characterized for their roles in mediating the regulatory effects of Gpa2 in mitochondria.